acidified with 5 M HCl and extracted with EtOAc. The organic
phase were combined and subjected to silica gel column
chromatography to yield the compound 2P as white solid. 1H
NMR (400 MHz, CD3OD) δ 7.27 (d, J = 2.0 Hz, 1H), 7.00 (d, J
= 2.0 Hz, 1H), 3.85 (s, 3H), 2.54 (t, J = 7.4 Hz, 2H), 1.68-1.59
(m, 2H), 0.94 (t, J = 7.4 Hz, 3H); 13C NMR (100 MHz,
CD3OD) δ 176.1, 151.3, 149.4, 134.1, 122.0, 118.7, 114.1, 55.7,
38.5, 25.7, 14.0.
4-Hydroxy-5-methoxyisophthalic acid (3P): Following the
general procedure outlined above for compound 2P. Compound
3P were obtained as white solid. 1H NMR (400 MHz, DMSO-
D6) δ 12.6 (br, s, 1H), 8.01 (d, J = 1.9 Hz, 1H), 7.60 (d, J = 1.9
Hz, 1H), 3.85 (s, 3H); 13C NMR (100 MHz, DMSO-D6) δ
171.8, 166.5, 155.4, 148.1, 123.5, 120.9, 116.1, 112.9, 55.9.
HRMS: Calculated for C9H9O6 ([M+H]+): 213.0399; found:
213.0400.
min with H2O-formic acid (0.1%) as the mobile phase (flow
rate 1.0 mL/min) and an acetonotrile-formic acid (0.1%)
gradient (0-2 min 5%, 2-30 min 5-100%).
Determination of substrate conversion.
All analyses were carried on a HPLC system from DIONEX or
Elite HPLC equipped with a UV-detector and a reverse-phase
Agilent Zorbax SB-Aq, 250×4.6 mm, 5 µm, column
temperature 30 oC. Conversions were determined by
comparison with calibration curves for the substrate prepared
with authentic reference material. All compounds were detected
at 254 nm or 280 nm, respectively.
Method for 1, 1P, 2, 2P, 3, 3P, 5, 5P, 6, 6P, 8 and 8P was
run over 30 min with H2O-trifluoroactetic acid (0.1%) as the
mobile phase (flow rate 1.0 mL/min) and an acetonotrile-
trifluoroacetic acid (0.1%) gradient (0-2 min 5%, 2-30 min 5-
2-Hydroxy-3-methoxy-5-methylbenzoic acid (4P)27:
Following the general procedure outlined above for compound
2P. Compound 4P were obtained as white solid. 1H NMR (400
MHz, CDCl3) δ 10.36 (s, 1H), 7.31 (d, J = 1.6 Hz, 1H), 6.92 (d,
J = 1.6 Hz, 1H), 3.91 (s, 3H), 2.32 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 174.6, 150.5, 148.4, 128.6, 121.5, 119.1, 111.2,
56.4, 21.1.
100%). Retention times:
min, 2P 20.97 min, 13.51 min, 3P 14.69 min,
12.40 min, 6P 14.22 min, 8 13.85 min, 8P 14.69
1
21.25 min, 1P 19.89 min,
2 21.39
3
5
13.37 min, 5P
14.73 min,
min.
6
Method for
4 and 4P was run over 30 min with H2O-
trifluoroactetic acid (0.1%) as the mobile phase (flow rate 1.0
mL/min) and an acetonotrile-trifluoroacetic acid (0.1%)
gradient (0-2 min 5%, 2-20 min 5-50%, 20-30 min 50-100 %).
5-(2-Carboxyvinyl)-2-hydroxybenzoic acid (5P)24: Following
the general procedure outlined above for compound 2P
.
Retention times:
4 19.79 min, 4P 20.70 min.
Compound 5P were obtained as white solid (24.3%). 1H NMR
(400 MHz, DMSO-D6) δ 8.02 (d, J = 2.0 Hz, 1H), 7.89 (dd, J =
8.6, 2.0 Hz, 1H), 7.56 (d, J = 16.0 Hz, 1H), 6.99 (d, J = 8.6 Hz,
1H), 6.39 (d, J = 16.0 Hz, 1H); 13C NMR (100 MHz, DMSO-
D6) δ 171.5, 167.7, 162.6, 143.0, 134.5, 131.1, 125.6, 118.0,
117.4, 113.6.
Method for and 7P was run over 30 min with H2O-
7
trifluoroactetic acid (0.1%) as the mobile phase (flow rate 1.0
mL/min) and an acetonotrile-trifluoroacetic acid (0.1%)
gradient (0-2 min 5%, 2-23 min 5-32.5%, 23-30 min 32.5-
100 %). Retention times:
7 20.80 min, 7P 22.86 min.
Molecule simulation
5-(2-Carboxyethyl)-2-hydroxybenzoic acid (6P): Following
the general procedure outlined above for compound 2P
.
Molecule docking of either the substrates or the corresponding
products to LigW_Sp and LigW2_Sp active sites was
performed by AutoDock Vina (version 1.1.2). All structures of
substrates and products were constructed by ChemDraw Ultra
8.0, and prepared by the AutoDock Tools 1.5.6 (ADT). The
crystal structure of LigW_Sp was retrieved from the RCSB
Protein Data Bank (RCSB PDB) and chain A of 4L6D (PDB
code) was used as target for molecule docking assays. The
crystal structure of LigW2_Sp was homology modeled using
the SWISS-MODEL server by choosing chain A of crystal
structure of 5-carboxyvanillate decarboxylase LigW2 from
Novosphingobium aromaticivorans DSM 12444 (RCSB PDB
code: 4QS5) as template. All the proteins were prepared by the
ADT, including removing water molecules and the native
ligands, adding hydrogens. The substrates and corresponding
products were docked into the active site of LigW_Sp or
LigW2_Sp using the native ligands position. All docking assays
were performed at least five times. The final structures and
locations of either substrates or products on the target proteins
were identified based on the favorable RMSD and binding
energy. All graphic presentations of structures were generated
using PyMOL.
Compound 6P were obtained as white solid (24.1%). 1H NMR
(400 MHz, CD3OD) δ 7.71 (d, J = 2.2 Hz, 1H), 7.33 (dd, J =
8.5, 2.2 Hz, 1H), 6.84 (d, J = 8.5 Hz, 1H), 5.13 (br, 1H), 2.84 (t,
J = 7.5 Hz, 2H), 2.56 (t, J = 7.5 Hz, 2H); 13C NMR (100 MHz,
CD3OD) δ 176.6, 173.4, 161.6, 136.8, 132.7, 130.8, 118.2,
113.6, 36.7, 30.9. (Data is consistent with the literature)24
5-(2-Carboxyvinyl)-2-hydroxy-3-methoxybenzoic acid (7P):
Following the general procedure outlined above for compound
2P. Compound 7P were obtained as white solid. 1H NMR (400
MHz, DMSO-D6) δ 7.58 (d, J = 1.6 Hz, 1H), 7.55 (d, J = 1.6
Hz, 1H), 7.52 (d, J = 15.9 Hz, 1H), 6.48 (d, J = 15.9 Hz, 1H),
3.86 (s, 3H); 13C NMR (100 MHz, DMSO-D6) δ 172.0, 167.8,
153.6, 148.7, 143.5, 125.1, 123.0, 117.6, 114.5, 113.1, 56.1.
HRMS: Calculated for C11H11O6 ([M+H]+): 239.0556; found:
239.0548.
5-(2-Carboxyethyl)-2-hydroxy-3-methoxybenzoic acid (8P):
Following the general procedure outlined above for compound
2P. Compound 8P were obtained as white solid. 1H NMR (400
MHz, CD3OD) δ 7.31 (d, J = 1.9 Hz, 1H), 7.05 (d, J = 1.9 Hz,
1H), 3.85 (s, 3H), 2.85 (t, J = 7.5 Hz, 2H), 2.59 (t, J = 7.5 Hz,
2H); 13C NMR (100 MHz, CD3OD) δ 176.6, 173.8, 151.7,
149.5, 132.5, 121.9, 118.6, 113.9, 56.7, 36.7, 31.5; HRMS:
Calculated for C11H13O6 ([M+H]+): 241.0712; found: 241.0706.
Data Availability
Analytical procedures
Additional data of this work have been uploaded as the electronic
supplementary material. Figure S1: SDS-PAGE results of
overexpressed (de)carboxylases in E. coli BL21 (DE3), Figure S2:
Docking results of substrates into the active sites of LigW_Sp or
General. HPLC-MS experiments were acquired on Agilent
6540 QTOF and a reversed phase Agilent Zorbax SB-Aq,
250×4.6 mm, 5 µm, at 30 oC. Method for 1P was run over 30
5